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Pseudorhizobium pelagicum gen. nov., sp. nov. isolated from a pelagic Mediterranean zone
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Pseudorhizobium pelagicum gen. nov., sp. nov. isolated from a pelagic Mediterranean zone夽 Nikole E. Kimes a , Mario López-Pérez a , José David Flores-Félix b , Martha-Helena Ramírez-Bahena c,d , José M. Igual c,d , Alvaro Peix c,d , Francisco Rodriguez-Valera a , Encarna Velázquez b,d,∗ a
Evolutionary Genomics Group, División de Microbiología, Universidad Miguel Hernández, Apartado 18, San Juan 03550, Alicante, Spain Departamento de Microbiología y Genética. Universidad de Salamanca, Salamanca, Spain c Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de Investigaciones Científicas (IRNASA-CSIC), Salamanca, Spain d Unidad Asociada Grupo de Interacción Planta-Microorganismo Universidad de Salamanca-IRNASA-CSIC, Salamanca, Spain b
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 15 May 2015 Accepted 19 May 2015 Keywords: Rhizobiaceae Pseudorhizobium Taxonomy Sea Spain
a b s t r a c t Two novel Alphaproteobacteria strains, R1-200B4T and R2-400B4, were isolated from the Mediterranean Sea off the coast of Alicante in Spain. The phylogenetic analysis of the 16S rRNA gene showed that they are related to members of Family Rhizobiaceae. The 16S rRNA gene sequence of strain R1-200B4T presents 97.0% and 95.6% similarity with respect to the type strains of the type species from genera Neorhizobium and Rhizobium, Neorhizobium galegae HAMBI 540T and Rhizobium leguminosarum USDA 2370T , respectively. The remaining genera of family Rhizobiaceae showed similarities lower than 95%. The recA and atpD gene sequences of strain R1-200B4T showed, respectively, 90% and 88.6% similarity with respect to N. galegae HAMBI 540T and 87% and 86% with respect to R. leguminosarum USDA 2370T . The calculated ANI values between the genomes of the strain R1-200B4T and those of N. galegae HAMBI 540T and R. leguminosarum 3841 are 75.9% and 74.0%, respectively. The major fatty acids are those from summed feature 8 (C18:1 6c/C18:1 7c) and the C16:0 . Catalase and oxidase were positive. Nitrate reduction and aesculin hydrolysis were positive. Production of -galactosidase and urease was positive. The production of indol, arginine dehydrolase or gelatinase was negative. Growth was observed in presence of 7% NaCl. Therefore, based on the phylogenetic, chemotaxonomic and phenotypic data obtained in this study, we propose to classify the strains isolated in this study in a new genus named Pseudorhizobium gen. nov. and a new species named Pseudorhizobium pelagicum sp. nov. with the type strain R1-200B4T (= LMG 28314T = CECT 8629T ). © 2015 Published by Elsevier GmbH.
The Family Rhizobiaceae from alpha Proteobacteria currently encompasses eight genera, namely Rhizobium [9,32], Ensifer [6], Shinella [2], Ciceribacter [10], Neorhizobium [16], Agrobacterium, Allorhizobium and Pararhizobium [17]. Some of these genera are phylogenetically close with 16S rRNA gene similarity around 97%, as occurs in the case of genera Ciceribacter and Ensifer [10]. Similar values are also commonly found among genera of other families from the order Rhizobiales, such as Phyllobacteriaceae and Bradyrhizobiaceae, which are phylogenetically related to family
夽 Whole genome shotgun sequence: JOKI01000001. ∗ Corresponding author at: Departamento de Microbiología y Genética, Edificio Departamental de Biología, Universidad de Salamanca, Lab 209, Doctores de la Reina s/n, Salamanca 37007, Spain. Tel.: +34 923 294532; fax: +34 923 224876. E-mail address: [email protected] (E. Velázquez).
Rhizobiaceae. Within Phyllobacteriaceae more than 97% similarity in the 16S rRNA gene sequences is found between Aminobacter and Mesorhizobium and between Aquamicrobium and Defluvibacter. Within the family Bradyrhizobiaceae similarity values even higher than 97% are found among the genera Oligrotropha, Afipia, Nitrobacter, Metalliresistens, Rhodopseudomonas, Tardiphaga, and Bradyrhizobium (see Fig. 1). Hence, 16S rRNA similarity values around 97% is the current limit for delineation of genera in several families from the order Rhizobiales. Members of the family Rhizobiaceae are commonly involved in plant-microbe interactions; however, some of them have been isolated from other sources, although there are no marine isolates within this family [18]. Therefore, the taxon described in this work represents the first marine member of family Rhizobiaceae. The genetic and phenotypic characteristics support its classification into a new genus and a new species for which the names
http://dx.doi.org/10.1016/j.syapm.2015.05.003 0723-2020/© 2015 Published by Elsevier GmbH.
Please cite this article in press as: N.E. Kimes, et al., Pseudorhizobium pelagicum gen. nov., sp. nov. isolated from a pelagic Mediterranean zone, Syst. Appl. Microbiol. (2015), http://dx.doi.org/10.1016/j.syapm.2015.05.003
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94
Allorhizobium undicola LMG 11875T (Y17047) Agrobacterium radiobacter ATCC 19358T (AJ389904)
60
Shinella granuli Ch06T (AB187585) Rhizobium leguminosarum USDA 2370T (JQ085246) 80
64
Neorhizobium galegae HAMBI 540T (HG938353) Pseudorhizobium pelagicum 99 Ensifer adhaerens LMG 20216T (AM181733)
R1-200B4T
Family Rhizobiaceae
(JOKI01000001)
Ciceribacter lividus MSSRFBL1T (JQ230000)
63
Pararhizobium giardinii H152T (U86344) Hoeflea marina LMG 128T (AY598817) Phyllobacterium myrsinacearum IAM 13584T (D12789) Mesorhizobium loti NZP 2213T (KM192337) Aminobacter aminovorans DSM 7048T (AJ011759)
72
Nitratireductor aquibiodomus NL21T (NR025262) Pseudaminobacter salicylatoxidans KTC001T (AJ294416) Aquamicrobium defluvium LMG 22048T (Y15403) 99
99
Family Phyllobacteriaceae
Defluvibacter lusatiensis DSM 11099T (AJ132378) Chelativorans multitrophicus DSM 9103T (EF457243)
Aliihoeflea aestuarii N8T (EF660756) 97
Pseudahrensia aquimaris HDW-32T (GU575117)
97
Salinarimonas rosea YIMYD3T (EU878006) Balneimonas flocculans ATCC BAA-817T (AB098515) Bosea thiooxidans DSM 9653T (AJ250796)
100
71
Rhodoblastus acidophilus DSM 137T (FR733696)
74
Oligotropha carboxidovorans OM5T (CP001196) Afipia felis ATCC 53690T (M65248)
Family Bradyrhizobiaceae
Tardiphaga robiniae LMG 26467T (FR753034)
100
Nitrobacter winogradskyi Nb-255T (CP000115) Metalliresistens boonkerdii NS23T (EU177512 ) Bradyrhizobium japonicum USDA 6T (AB231927) 56
Rhodopseudomonas palustris ATCC 17001T (AB498815) Acetobacter aceti NCIB 8621T (X74066)
0.02 Fig. 1. Neighbor-joining plylogenetic tree based on 16S rRNA gene sequences (1317 positions) showing the relationships between Pseudorhizobium pelagicum gen. nov. sp. nov., the remaining genera from family Rhizobiaceae and the genera of the related families Phyllobacteriaceae and Bradyrhizobiaceae. The significance of each branch is indicated by a bootstrap value (in percentage) calculated for 1000 subsets (only values higher than 50% are indicated). Bar, 2 substitution per 100 nucleotide position.
Pseudorhizobium gen. nov. and Pseudorhizobium pelagicum sp. nov. are proposed, respectively. In this work two strains, R1-200B4T and R2-400B4, were isolated from Mediterranean seawater that was collected off the coast of Alicante, Spain (GPS coordinates = 38◦ 16 842N/00◦ 14 398W). Using a Niskin water sampler, 3 l of seawater were procured from 50 m depth, where the water temperature was 16 ◦ C. The seawater was placed in autoclaved pyrex bottles using sterile technique, covered from light, and kept cool with ice packs during the transport back to the laboratory (∼1.5 h). In the laboratory, 50 ml of the seawater was filtered using a 0.22 m syringe filter (Millipore Co., USA) and subsequently 200 l of a suspension eluted from the filter was plated on each of 10 minimal media agarose plates with antibiotics (MMAab: filtered and autoclaved seawater, 100 g/ml cycloheximide, 25 g/ml nalidixic acid, and 1% agarose). The plates were then incubated at 16 ◦ C with 12 h light/dark cycles for eight weeks. After eight weeks, colonies were streaked on marine agar (MA: 3.5% sea salt, 0.5% peptone, 0.1% yeast extract, 1.5% agar) plates and incubated at room temperature for 48 h. Genomic DNA was extracted from the colonies after they were grown in marine broth for 48 h, and the cells pelleted using centrifugation at 8650 × g for 10 min. After resuspending the cell pellet in lysis buffer (40 mM EDTA, 50 mM Tris pH = 8.3, and 0.75 M sucrose) with lysozyme (1 mg/ml), the solution was incubated at 37 ◦ C for 45 min. Then proteinase K (0.2 mg/ml) and SDS (0.1%) were added, and the solution incubated at 55 ◦ C for 1 h. Subsequently, the entire
solution was phenol/chloroform extracted twice, using an equal amount of phenol/chloroform/isoamyl alcohol (25:24:1 pH = 8) to that of the cell pellet solution. After centrifugation at 6182 × g for 10 min, the resulting supernatant was added in equal volume to chloroform/isoamyl alcohol (24:1) and again centrifuged at 6182 × g for 10 min. The genomic DNA was finally concentrated using ethanol precipitation, and the quality assessed using a spectrophotometer and gel electrophoresis. The genomic DNA (5 g) was sequenced using an Illumina HiSeq 2500 platform with 100 bp paired end reads at GATC Biotech (Constance, Germany). The resulting sequences (∼2 Gb per isolate) were preprocessed to eliminate low-quality bases and reads shorter than 50 bp. The trimmed sequences were assembled de novo using IDBA 1.1.1. (http://i.cs.hku.hk/∼alse/ hkubrg/projects/idba/) and Geneious (http://www.geneious.com/ ). The genomes were annotated using the NCBI PGAAP annotation pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation prok/ ). The complete 16S rRNA gene and housekeeping genes (recA and atpD) were identified in the annotated sequences and compared to the nonredundant NCBI database using BLASTN and BLASTP. The P. pelagicum R1-200B4T and P. pelagicum R2-400B4 genomes have been deposited in the NCBI database under the bioprojects PRJNA252589 (accession number JOKI00000000.1) and PRJNA252590 (accession number JOKJ00000000.1), respectively. Genomic comparisons between R1-200B4T and R2-400B4 revealed that these two isolates were very closely related, yet
Please cite this article in press as: N.E. Kimes, et al., Pseudorhizobium pelagicum gen. nov., sp. nov. isolated from a pelagic Mediterranean zone, Syst. Appl. Microbiol. (2015), http://dx.doi.org/10.1016/j.syapm.2015.05.003
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Pseudorhizobium pelagicum R1-200B4T (JOKI01000001) Neorhizobium galegae HAMBI 450T (HG938353) Shinella granuli CCUG 56671T (FM999739, FM999743) Ciceribacter lividus MSSRFBL1T (DSM 25528T) (KC189949, KP898900)
56
Ensifer adhaerens LMG 20216T (AJ505595, AM418746) Pararhizobium giardinii H152T (HQ394251, HQ394216)
58
Family Rhizobiaceae
Allorhizobium undicola LMG 11875T (EF457952, AM418784)
65
Agrobacterium radiobacter NCBI 9042T (ATCC 19358T) (HQ735079, HQ735092)
Rhizobium leguminosarum USDA 2370T (AJ294376, AJ294405) Phyllobacterium myrsinacearum ATCC 43590T (AJ294365, AJ294387) 51
90
Aminobacter aminovorans STM 2150 T (FR869646, AY785349) Pseudaminobacter salicylatoxidans KTC001T (NZ CAIU01000024)
60
Mesorhizobium loti LMG6125T (KM192346, KM192339) Aquamicrobium defluvii W13Z1 (|JENY01000009)
63 86
Family Phyllobacteriaceae
Nitratireductor aquibiodomus NL21T (NZ_BAMP01000152) 95
82
Balneimonas flocculans ATCC BAA-817T (JAEA01000006) Bosea thiooxidans LMG 26210T (FR871216, FR871211) Salinarimonas rosea DSM 21201T (NZ AUBC01000001) Nitrobacter winogradskyi Nb-255T (CP000115)
96
Rhodopseudomonas palustris HaA2 (CP000250)
99
Metalliresistens boonkerdii NS23T (EU177536, EU177520) 75
Oligotropha carboxidovorans OM5T (CP001196)
Family Bradyrhizobiaceae
Afipia felis ATCC 53690T (AGWZ01000001) Tardiphaga robiniae LMG 26467T (FR772688, FR873651) Bradyrhizobium japonicum USDA 6T (AP012206)
0.02 Fig. 2. Neighbor-joining phylogenetic tree based on partial concatenated sequences of recA and atpD genes (by this order and with a total of 820 positions) showing the relationships between Pseudorhizobium pelagicum gen. nov. sp. nov., the remaining genera from family Rhizobiaceae and the genera of the related families Phyllobacteriaceae and Bradyrhizobiaceae. The significance of each branch is indicated by a bootstrap value (in percentage) calculated for 1000 subsets (only values higher than 50% are indicated). Bar, 2 substitutions per 100 nucleotide positions.
distinct microorganisms. The analysis of nucleotide identity (ANI), based on whole genome sequencing and calculated using JSpecies software package v1.2.1 and default parameters [20], is high (99.98%) between the two strains over the core genome; however, coverage (98.37%) was not 100% as expected with clones of the same microorganism. This indicates that the genomes are not identical (i.e., clones) and contain genomic areas unique to one or the other strain. In fact, the R1-200B4T genome is slightly smaller (5.13 Mbp, 4539 CDS) compared to R2-400B4 (5.24 Mbp, 4619 CDS) with 168 unique genes between the two isolates. Most notably, R2400B4 contains a large lysogenic phage not present in R1-200B4T (Fig. S1). Furthermore, single nucleotide polymorphism (SNP) analysis between the two genomes revealed 1149 SNPs with a dN/dS ratio of 0.195, which provides additional evidence that these two isolates are not clones. Rather these data indicate that R1-200B4T and R2-400B4 belong to the same clonal frame, which describes bacterial lineages of common ancestry descent, in which replacement of genome fragments by recombination, selection, and drift by neutral genetic mutations has occurred [15]. In comparison, three non-clonal strains of Alteromonas macleodii comprising a single clonal frame (strains AltDE1, UM7/UM8, and UM4b) have a similarly high ANI (99.97%), yet exhibit only 87 SNPs and a maximum of 85 unique genes [14]. In addition to genomic comparisons, differences in antibiotic resistance (see below) and the RAPD patterns obtained with the primer M13 (5 -GAGGGTGGCGGTTCT-3 ) using the methodology described by Rivas et al. [21] confirmed that the two strains are not clones (Fig. S2). For comparison between the strains isolated in this study and their closest phylogenetic relatives, the 16S rRNA, recA and atpD gene sequences were identified from the R1-2004BT and R2-400B4 genomes. The 16S rRNA gene sequences of the two isolated strains were identical, thus only the sequence of the
type strain R1-200B4T was included in the phylogenetic analyses. Subsequent 16S rRNA, recA and atpD gene comparisons were made with those from GenBank using the BLASTN program [1] and the 16S rRNA gene sequences were also compared with those from EzTaxon-e server [11]. Sequences were aligned using the ClustalX software [30]. The distances were calculated according to Kimura’s two-parameter model [12]. Phylogenetic trees were inferred using the neighbour-joining (NJ) [24] and maximum likelihood (ML) [22] analyses. MEGA5 software [29] was used for all analyses. According to the results of comparison against the sequences held in EzTaxon-e server, the 16S rRNA gene sequence of strain R1-200B4T presents 97.0% and 95.6% similarity with respect to the type strains of the type species from genera Neorhizobium and Rhizobium, Neorhizobium galegae HAMBI 540T and Rhizobium leguminosarum USDA 2370T , respectively. The remaining genera of family Rhizobiaceae showed similarities lower than 95%. The NJ and ML phylogenetic analyses (Fig. 1 and Fig. S3, respectively) confirmed these results showing that the distance found between the strain R1-200B4T and N. galegae HAMBI 540T is similar or higher than those found among some species of family Phyllobacteriaceae previously mentioned in this work and among most of genera from Bradyrhizobiaceae. The recA and atpD genes have been sequenced in the type strains of the type species from all genera of family Rhizobiaceae and in most of type species of the families Phyllobacteriaceae and Bradyrhizobiaceae. Although for some species these sequences are available for strains other than the type they were included in this work. Since the sequences of recA and atpD genes are identical in the strains from this study, only the genes of the type strain R1-200B4T were included in the NJ and ML phylogenetic analyses that yielded phylogenetic trees with similar topology (Fig. 2 and Fig. S4, respectively). These analyses showed that strain R1-200B4T clustered with its
Please cite this article in press as: N.E. Kimes, et al., Pseudorhizobium pelagicum gen. nov., sp. nov. isolated from a pelagic Mediterranean zone, Syst. Appl. Microbiol. (2015), http://dx.doi.org/10.1016/j.syapm.2015.05.003
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Table 1A Calculated ANI values for available genomes of strains from type species of different genera from Family Rhizobiaceae. The accession numbers for the genomes of Pseudorhizobium pelagicum R1-200B4T , Neorhizobium galegae HAMBI 540T , Rhizobium leguminosarum 3841 and Ensifer adhaerens strain AT were, respectively, JOKI01000001, HG938353, AM236080 and NZ JNAE00000000, GCA 000421945, NZ JHXQ00000000, NZ ARBG00000000. Species
P. pelagicum R1-200B4T
N. galegae HAMBI 540T
R. leguminosarum 3841
E. adhaerens strain AT
A. undicola ORS 992T
A. radiobacter DSM 30147T
P. giardinii H152T
P. pelagicum R1-200B4T N. galegae HAMBI 540T R. leguminosarum 3841 E. adhaerens strain AT A. undicola ORS 992T A. radiobacter DSM 30147T P. giardinii H152T
100 75.9 74.0 73.6 72.4 73.8 73.2
100 75.1 73.3 72.6 73.6 73.5
100 74.0 72.7 73.3 74.6
100 73.0 72.6 76.1
100 72.7 72.2
100 72.6
100
closest relative N. galegae HAMBI 540T with 90% and 88.6% similarity values, respectively. The type strain of R. leguminosarum USDA 2370T was more distant with 87% and 86% similarity values in recA and atpD genes, respectively. However, similar values to those found between strain R1-200B4T and N. galegae HAMBI 540T were also found between R1-200B4T and Shinella granuli CCUG56671T with 90% and 88.8% similarity, respectively. These values are equal or lower than those presented by other members of Family Rhizobiaceae such as Ensifer adhaerens ATCC 33212T or S. granuli CCUG56671T and the recently described Ciceribacter lividus MSSRFBL1T with 90% and 93% similarity values, respectively, in recA and atpD genes. Also similar values, 91% and 93%, respectively, are found between E. adhaerens ATCC 33212T or S. granuli CCUG56671T . In families Phyllobacteriaceae and Bradyrhizobiaceae the closest related genera for which recA and atpD genes are availale presented similarity values near to 90% in both genes, as occurs between Aminobacter aminovorans STM 2150T and Pseudaminobacter salicylatoxidans KCT001T from family Phyllobacteriaceae or between Oligotropha carboxidovorans OM5T and Afipia felis ATCC 53690T from family Bradyrhizobiaceae. These values support those obtained from 16S rRNA gene analysis showing that the strain R1-200B4T belongs to a new genus within the family Rhizobiaceae. ANI values are commonly used to delineate species, but they have also been evaluated for genera delineation in a recent work where several new genera have been proposed finding that ANI values ranged from 75 to 80% among genera of the family Enterobacteriaceae [28]. The calculated ANI values between the genome of strain R1-200B4T and those of strains from family Rhizobiaceae available in databanks are in Tables 1A–1C. All of them are similar or lower than those found among genera of family Enterobacteriaceae. The calculated ANI value between the genomes of the strains R1-200B4T and N. galegae HAMBI 540T is 75.9%. This value is similar to the ANI values found between the strain N. galegae HAMBI 540T and R. etli CFN42T . These findings also support the classification of strains R1-200B4T and R2-400B4 in a new genus. The G + C content was calculated from the sequenced genome data being 62.8% for the strain R1-200B4T and 62.4% for the strain R2-400B4. This value is similar to those found in the remaining genera of Family Rhizobiaceae [13]. The cellular fatty acids were analysed by using the Microbial Identification System (MIDI; Microbial ID) Sherlock 6.1 and the library RTSBA6 according to the technical instructions provided by this system [25]. The strains were cultured aerobically on TY plates
[4] at 28 ◦ C and cells were collected during the late-exponential phase of growth. The obtained results showed that the major fatty acids of P. pelagicum are those from summed feature 8 (C18:1 6c/C18:1 7c) and the C16:0 , followed by C19:0 cyclo 8c, C8:1 7c 11-methyl, C18:0 , summed feature 3 (C16:1 6c/C16:1 7c) and summed feature 2 (C12:0 aldehyde?/C14:0 3OH/C16:1 iso I). Although some differences were found in the proportions of some fatty acids among genera from Family Rhizobiaceae (Table 2), the major fatty acids of this new genus coincide with those found in the remaining genera of the family Rhizobiaceae and also with those of families Phyllobacteriaceae and Bradyrhizobiaceae [31]. The phenotypic characterization was performed in this study using the methodology previously reported [19,23]. The type strains of the type species for all the defined genera in Family Rhizobiaceae were included in the phenotypic study as reference (Table 3). Phenotypic characteristics of the new species are reported below in the species description and the differences with respect to the type species of the genera from family Rhizobiaceae are recorded in Table 3. The two strains from this study differ in their natural antibiotic resistance; the strain R1-200B4T was sensitive to cefuroxime, whereas the strain R2-400B4 was resistant. A common characteristic of species from genus Rhizobium is their ability to form symbiotic relationships with plants, which in turn results in biological nitrogen fixation. This symbiosis requires, among others, three major genetic components: nodulation (nod), nitrogenase (nif) and nitrogen fixation (fix) genes. Initially nod genes are crucial in inducing communication factors and establishing symbiosis with the host [8]. Subsequently nitrogenase, an enzyme complex encoded by nifHDK genes, catalyzes nitrogen fixation with the assistance of more than 15 additional nif genes [27]. Since nitrogen fixation is an energy-consuming process, effective symbiosis also depends on the operation of a respiratory chain with a high affinity for O2 , closely coupled to ATP production. Under these circumstances, the regulation of oxygen is controlled by the fix gene family that codes for a special three-subunit terminal oxidase (cytochrome terminal oxidase, cbb3) and a membrane complex participating in electron transfer to nitrogenase [5]. In the case of R1-200B4T and R2-400B4, the genomes show no evidence of nod genes, suggesting that these two strains are free-living microorganisms that do not form symbiotic associations unlike many of their closest relatives. In addition, the absence of nif genes further indicates that these microorganisms do not perform nitrogen fixation, which requires nitrogenase. Although rhizobia
Table 1B Calculated ANI values for available genomes of strains from type species of different genera from Family Bradyrhizobiaceae. The accession numbers for the genomes of Bradyrhizobium japonicum USDA 6T , Afipia felis ATCC 53690T , Nitrobacter winogradskyi Nb-255T and Rhodopseudomonas palustris HaA2 were, respectively, NC 017249, NZ AGWZ00000000, NC 007406 and NC 007778. Species
B. japonicum USDA 6T
A. felis ATCC 53690T
N. winogradskyi Nb-25T
R. palustris HaA2
B. japonicum USDA 6T A. felis ATCC 53690T N. winogradskyi Nb-255 R. palustris HaA2
100 74.0 75.8 76.3
100 74.2 74.3
100 76.2
100
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Table 1C Calculated ANI values for available genomes of strains from type species of different genera from Family Phyllobacteriaceae. The accession numbers for the genomes of Mesorhizobium loti MAFF303099, Pseudaminobacter salicylatoxidans KCT001, Aquamicrobium defluvii W13Z1 were, respectively, NC 002678, NZ JAGL00000000, JENY00000000 and NZ BAMP00000000. Species
M. loti MAFF303099
P. salicylatoxidans KCT001
A. defluvii W13Z1
N. aquibiodomus NL21T
M. loti MAFF303099 P. salicylatoxidans KCT001 A. defluvii W13Z1 N. aquibiodomus NL21T
100 75.3 75.7 72.1
100 75.3 72.5
100 72.1
100
Table 2 Cellular fatty acid composition of the type species from genera of family Rhizobiaceae. Data are from this study. Fatty acids present in amounts lower than 1% are not shown. nd. not detected. Data are from this study. Fatty acid
P. pelagicum R1-200B4T
N. galegae HAMBI 540T
R. leguminosarum USDA 2370T
C. lividus DSM 25528T
E. adhaerens LMG 20216T
S. granuli DSM 18401T
A. radiobacter ATCC 19358T
A. undicola LMG 11875T
P. giardinii H152T
C16:0 C17:0 C18:0 C16:0 3OH C18:0 3OH C18:1 7c 11-methyl C19:0 cyclo 8c Summed feature 2 Summed feature 3 Summed feature 8
7.69 nd 2.26 nd 1.49 3.04 4.37 4.41 1.49 73.84
11.58 1.91 2.28 2.54 1.60 0.55 10.76 4.63 0.63 61.14
7.18 nd 12.71 nd 0.99 7.06 3.74 3.59 1.19 62.77
3.72 nd 8.88 nd 1.50 nd 8.06 3.13 nd 74.71
2.89 nd 4.05 1.75 2.52 2.09 0.42 6.03 0.94 79.31
10.68 nd 1.71 1.65 0.62 10.14 18.30 3.51 1.70 50.55
8.64 nd 1.08 4.82 0.42 0.37 2.86 6.10 2.06 73.64
0.88 nd 0.83 2.48 1.35 1.54 1.22 5.65 2.98 82.01
6.57 nd 0.62 nd 2.26 nd nd 8.77 6.89 74.89
Summed feature 2: (C14:0 3OH/C16:1 iso I/C12:0 aldehyde?). Summed feature 3: (C16:1 7c/C16:1 6c). Summed feature 8: (C18:1 7c/C18:1 6c).
are most commonly associated with plant symbiosis, it has been shown in numerous examples that this is not always the case [7]. Even within a given species, such as R. leguminosarum, some strains can lack the ability to form symbiotic relationships, while others are common symbiotic microorganisms [26]. More recently, Rhizobium sp. NT-26, isolated from a contaminated acid mine, was shown not to form nodules and lacked both nodulation and nitrogen fixation genes [3]. Nevertheless, as most genera of family Rhizobiaceae contain symbiotic and non-symbiotic species and it is possible that future species of the new genus Pseudorhizobium can be able to form nitrogen-fixing nodules in legumes, we have not included these characteristics in the genus or species descriptions.
Based on the phenotypic, chemotaxonomic and genotypic characteristics we propose to classify the studied strains into a novel genus named Pseudorhizobium gen. nov. and to a new species named P. pelagicum sp. nov. Description of genus Pseudorhizobium gen. nov. Pseudorhizobium (Gr. adj. pseudes false; N.L. masc. n. Pseudorhizobium, false Rhizobium). Aerobic, Gram negative non-spore forming rods forming white colonies on YMA. Optimal growth at 28 ◦ C and pH7. Catalase and
Table 3 Differential phenotypic characteristics of P. pelagicum and the type species from the remaining genera of family Rhizobiaceae. Data are from this study. +: positive, −: negative, w: weak. Characteristics
P. pelagicum R1-200B4T
N. galegae HAMBI 540T
R. leguminosarum USDA 2370T
C. lividus DSM 25528T
E. adhaerens LMG 20216T
S. granuli DSM 18401T
A. radiobacter ATCC 19358T
A. undicola LMG 11875T
P. giardinii H152T
Colony colour (on YMA)
White
Bluish black − + − +
Ivory
+ + + +
White– cream − − − −
White
Nitrate reduction Growth in 1% NaCl Growth in 7% NaCl Growth at 37 ◦ C
White– pink − + − +
+ + − +
+ + − +
White– cream + + − +
White– cream − + − +
White– cream − + − w
Assimilation of (API 20NE) Gluconate Malate
+ −
+ +
+ w
− +
− +
+ +
+ +
− +
− +
Assimilation of (API 32GN) l-Rhamnose d-Ribose Inositol Melibiose 5-Keto-gluconate 2-Keto-gluconate Propionate 3-Hydroxybutyrate l-Histidine l-Alanine l-Serine
+ − − − + w − − − w +
− + + + + − − − + + −
+ + + + − − − + + − −
+ + + − − − − − − − −
+ + + + − − + − + + +
+ + + − − − + + + + +
+ + + + w + + w + + −
+ w + − − − − − + + −
+ + + + − − − − + − −
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oxidase were positive. Nitrate reduction and aesculin hydrolysis were positive. Production of -galactosidase and urease was positive. The production of indol, arginine dehydrolase or gelatinase was negative. Growth was observed in presence of 7% NaCl. The main fatty acids are C18:1 7c/C18:1 6c. The G + C content of genomic DNA of the type strain of the type species is 62.8 mol%. Delineation of the genus was determined by the phylogenetic information from 16S rRNA gene sequence and by the analysis of the complete genome sequence. The type species is P. pelagicum. Description of Pseudorhizobium pelagicum sp. nov. P. pelagicum (pe.la.gi’cum. L. neut. adj. pelagicum of or belonging to the sea). Aerobic, Gram negative non-spore forming rods forming white colonies on YMA at 28 ◦ C that is the optimal growth temperature. Oxidase and catalase positive. Growth was obtained in a range from 10 to 37 ◦ C and pH 6 to 8. Weak growth observed at 40 ◦ C. The optimum pH is 7–7.5. No growth was observed at pH 4.5. Growth was observed in presence of 7% (w/v) NaCl. In API 20NE system nitrate reduction and aesculin hydrolysis were positive. Production of -galactosidase and urease was positive. The production of indol, arginine dehydrolase or gelatinase was negative. Glucose, l-arabinose, mannose, mannitol and gluconate were assimilated, but N-acetyl-glucosamine, malate, caproate, adipate, citrate and phenylacetate did not. The assimilation of maltose was weak. In API 32GN system the assimilation of l-rhamnose, N-acetyl-glucosamine, l-ribose, sucrose, maltose, mannitol, glucose, l-fucose, l-sorbose, l-arabinose, acetate, d,llactate, 5 keto-gluconate, l-serine and l-proline was positive. That of salicine, l-alanine, l-histidine, 2 keto-gluconate was weak. Inositol, melibiose, itaconate, suberate, malonate, glycogen, 3-hydroxybenzoate, 4-hydroxybenzoate, 3-hydroxybutyrate, propionate, caprate, valerate and citrate was negative. Sensitive to ciprofloxacin, oxitetracycline and gentamycin. Sensitive or weakly sensitive to neomycin. Resistant or weakly sensitive polymyxin B. Resistant to penicillin, ampicillin, erythromycin, and cloxacillin. Resistance to cefuroxime was variable. The fatty acid profile consisted of C18:1 6c/C18:1 7c (summed feature 8), C16:0 , C19:0 cyclo 8c, C8:1 7c 11-methyl, C18:0 , C16:1 6c/C16:1 7c (summed feature 3) and C12:0 aldehyde?/C14:0 3OH/C16:1 iso I (summed feature 2). The G + C content of the strain R1-200B4T is 62.8 mol%. No nodulation genes were found in the genome of the strains from this species. The type strain R1-200B4T (= LMG 28314T = CECT 8629T ) was isolated from Mediterranean seawater that was collected off the coast of Alicante, Spain. Acknowledgements This work was supported by funds from Junta de Castilla y León (Regional Spanish Government), MICINN (Central Spanish Government). “The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013.) under grant agreement no. 311975 for the MaCuMBA (http://www.macumbaproject.eu/) project”. MHRB is recipient of a JAE-Doc researcher contract from CSIC cofinanced by ERDF. JDFF is recipient of a PhD fellowship from Universidad de Salamanca. We thank Dr. J. Euzeby by his valuable help in provinding the correct ethymology for the name of the new taxon. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2004.08. 011
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Pseudorhizobium pelagicum gen. nov., sp. nov. isolated from a pelagic Mediterranean zone夽 Nikole E. Kimes a , Mario López-Pérez a , José David Flores-Félix b , Martha-Helena Ramírez-Bahena c,d , José M. Igual c,d , Alvaro Peix c,d , Francisco Rodriguez-Valera a , Encarna Velázquez b,d,∗ a
Evolutionary Genomics Group, División de Microbiología, Universidad Miguel Hernández, Apartado 18, San Juan 03550, Alicante, Spain Departamento de Microbiología y Genética. Universidad de Salamanca, Salamanca, Spain c Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de Investigaciones Científicas (IRNASA-CSIC), Salamanca, Spain d Unidad Asociada Grupo de Interacción Planta-Microorganismo Universidad de Salamanca-IRNASA-CSIC, Salamanca, Spain b
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 15 May 2015 Accepted 19 May 2015 Keywords: Rhizobiaceae Pseudorhizobium Taxonomy Sea Spain
a b s t r a c t Two novel Alphaproteobacteria strains, R1-200B4T and R2-400B4, were isolated from the Mediterranean Sea off the coast of Alicante in Spain. The phylogenetic analysis of the 16S rRNA gene showed that they are related to members of Family Rhizobiaceae. The 16S rRNA gene sequence of strain R1-200B4T presents 97.0% and 95.6% similarity with respect to the type strains of the type species from genera Neorhizobium and Rhizobium, Neorhizobium galegae HAMBI 540T and Rhizobium leguminosarum USDA 2370T , respectively. The remaining genera of family Rhizobiaceae showed similarities lower than 95%. The recA and atpD gene sequences of strain R1-200B4T showed, respectively, 90% and 88.6% similarity with respect to N. galegae HAMBI 540T and 87% and 86% with respect to R. leguminosarum USDA 2370T . The calculated ANI values between the genomes of the strain R1-200B4T and those of N. galegae HAMBI 540T and R. leguminosarum 3841 are 75.9% and 74.0%, respectively. The major fatty acids are those from summed feature 8 (C18:1 6c/C18:1 7c) and the C16:0 . Catalase and oxidase were positive. Nitrate reduction and aesculin hydrolysis were positive. Production of -galactosidase and urease was positive. The production of indol, arginine dehydrolase or gelatinase was negative. Growth was observed in presence of 7% NaCl. Therefore, based on the phylogenetic, chemotaxonomic and phenotypic data obtained in this study, we propose to classify the strains isolated in this study in a new genus named Pseudorhizobium gen. nov. and a new species named Pseudorhizobium pelagicum sp. nov. with the type strain R1-200B4T (= LMG 28314T = CECT 8629T ). © 2015 Published by Elsevier GmbH.
The Family Rhizobiaceae from alpha Proteobacteria currently encompasses eight genera, namely Rhizobium [9,32], Ensifer [6], Shinella [2], Ciceribacter [10], Neorhizobium [16], Agrobacterium, Allorhizobium and Pararhizobium [17]. Some of these genera are phylogenetically close with 16S rRNA gene similarity around 97%, as occurs in the case of genera Ciceribacter and Ensifer [10]. Similar values are also commonly found among genera of other families from the order Rhizobiales, such as Phyllobacteriaceae and Bradyrhizobiaceae, which are phylogenetically related to family
夽 Whole genome shotgun sequence: JOKI01000001. ∗ Corresponding author at: Departamento de Microbiología y Genética, Edificio Departamental de Biología, Universidad de Salamanca, Lab 209, Doctores de la Reina s/n, Salamanca 37007, Spain. Tel.: +34 923 294532; fax: +34 923 224876. E-mail address: [email protected] (E. Velázquez).
Rhizobiaceae. Within Phyllobacteriaceae more than 97% similarity in the 16S rRNA gene sequences is found between Aminobacter and Mesorhizobium and between Aquamicrobium and Defluvibacter. Within the family Bradyrhizobiaceae similarity values even higher than 97% are found among the genera Oligrotropha, Afipia, Nitrobacter, Metalliresistens, Rhodopseudomonas, Tardiphaga, and Bradyrhizobium (see Fig. 1). Hence, 16S rRNA similarity values around 97% is the current limit for delineation of genera in several families from the order Rhizobiales. Members of the family Rhizobiaceae are commonly involved in plant-microbe interactions; however, some of them have been isolated from other sources, although there are no marine isolates within this family [18]. Therefore, the taxon described in this work represents the first marine member of family Rhizobiaceae. The genetic and phenotypic characteristics support its classification into a new genus and a new species for which the names
http://dx.doi.org/10.1016/j.syapm.2015.05.003 0723-2020/© 2015 Published by Elsevier GmbH.
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Allorhizobium undicola LMG 11875T (Y17047) Agrobacterium radiobacter ATCC 19358T (AJ389904)
60
Shinella granuli Ch06T (AB187585) Rhizobium leguminosarum USDA 2370T (JQ085246) 80
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Neorhizobium galegae HAMBI 540T (HG938353) Pseudorhizobium pelagicum 99 Ensifer adhaerens LMG 20216T (AM181733)
R1-200B4T
Family Rhizobiaceae
(JOKI01000001)
Ciceribacter lividus MSSRFBL1T (JQ230000)
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Pararhizobium giardinii H152T (U86344) Hoeflea marina LMG 128T (AY598817) Phyllobacterium myrsinacearum IAM 13584T (D12789) Mesorhizobium loti NZP 2213T (KM192337) Aminobacter aminovorans DSM 7048T (AJ011759)
72
Nitratireductor aquibiodomus NL21T (NR025262) Pseudaminobacter salicylatoxidans KTC001T (AJ294416) Aquamicrobium defluvium LMG 22048T (Y15403) 99
99
Family Phyllobacteriaceae
Defluvibacter lusatiensis DSM 11099T (AJ132378) Chelativorans multitrophicus DSM 9103T (EF457243)
Aliihoeflea aestuarii N8T (EF660756) 97
Pseudahrensia aquimaris HDW-32T (GU575117)
97
Salinarimonas rosea YIMYD3T (EU878006) Balneimonas flocculans ATCC BAA-817T (AB098515) Bosea thiooxidans DSM 9653T (AJ250796)
100
71
Rhodoblastus acidophilus DSM 137T (FR733696)
74
Oligotropha carboxidovorans OM5T (CP001196) Afipia felis ATCC 53690T (M65248)
Family Bradyrhizobiaceae
Tardiphaga robiniae LMG 26467T (FR753034)
100
Nitrobacter winogradskyi Nb-255T (CP000115) Metalliresistens boonkerdii NS23T (EU177512 ) Bradyrhizobium japonicum USDA 6T (AB231927) 56
Rhodopseudomonas palustris ATCC 17001T (AB498815) Acetobacter aceti NCIB 8621T (X74066)
0.02 Fig. 1. Neighbor-joining plylogenetic tree based on 16S rRNA gene sequences (1317 positions) showing the relationships between Pseudorhizobium pelagicum gen. nov. sp. nov., the remaining genera from family Rhizobiaceae and the genera of the related families Phyllobacteriaceae and Bradyrhizobiaceae. The significance of each branch is indicated by a bootstrap value (in percentage) calculated for 1000 subsets (only values higher than 50% are indicated). Bar, 2 substitution per 100 nucleotide position.
Pseudorhizobium gen. nov. and Pseudorhizobium pelagicum sp. nov. are proposed, respectively. In this work two strains, R1-200B4T and R2-400B4, were isolated from Mediterranean seawater that was collected off the coast of Alicante, Spain (GPS coordinates = 38◦ 16 842N/00◦ 14 398W). Using a Niskin water sampler, 3 l of seawater were procured from 50 m depth, where the water temperature was 16 ◦ C. The seawater was placed in autoclaved pyrex bottles using sterile technique, covered from light, and kept cool with ice packs during the transport back to the laboratory (∼1.5 h). In the laboratory, 50 ml of the seawater was filtered using a 0.22 m syringe filter (Millipore Co., USA) and subsequently 200 l of a suspension eluted from the filter was plated on each of 10 minimal media agarose plates with antibiotics (MMAab: filtered and autoclaved seawater, 100 g/ml cycloheximide, 25 g/ml nalidixic acid, and 1% agarose). The plates were then incubated at 16 ◦ C with 12 h light/dark cycles for eight weeks. After eight weeks, colonies were streaked on marine agar (MA: 3.5% sea salt, 0.5% peptone, 0.1% yeast extract, 1.5% agar) plates and incubated at room temperature for 48 h. Genomic DNA was extracted from the colonies after they were grown in marine broth for 48 h, and the cells pelleted using centrifugation at 8650 × g for 10 min. After resuspending the cell pellet in lysis buffer (40 mM EDTA, 50 mM Tris pH = 8.3, and 0.75 M sucrose) with lysozyme (1 mg/ml), the solution was incubated at 37 ◦ C for 45 min. Then proteinase K (0.2 mg/ml) and SDS (0.1%) were added, and the solution incubated at 55 ◦ C for 1 h. Subsequently, the entire
solution was phenol/chloroform extracted twice, using an equal amount of phenol/chloroform/isoamyl alcohol (25:24:1 pH = 8) to that of the cell pellet solution. After centrifugation at 6182 × g for 10 min, the resulting supernatant was added in equal volume to chloroform/isoamyl alcohol (24:1) and again centrifuged at 6182 × g for 10 min. The genomic DNA was finally concentrated using ethanol precipitation, and the quality assessed using a spectrophotometer and gel electrophoresis. The genomic DNA (5 g) was sequenced using an Illumina HiSeq 2500 platform with 100 bp paired end reads at GATC Biotech (Constance, Germany). The resulting sequences (∼2 Gb per isolate) were preprocessed to eliminate low-quality bases and reads shorter than 50 bp. The trimmed sequences were assembled de novo using IDBA 1.1.1. (http://i.cs.hku.hk/∼alse/ hkubrg/projects/idba/) and Geneious (http://www.geneious.com/ ). The genomes were annotated using the NCBI PGAAP annotation pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation prok/ ). The complete 16S rRNA gene and housekeeping genes (recA and atpD) were identified in the annotated sequences and compared to the nonredundant NCBI database using BLASTN and BLASTP. The P. pelagicum R1-200B4T and P. pelagicum R2-400B4 genomes have been deposited in the NCBI database under the bioprojects PRJNA252589 (accession number JOKI00000000.1) and PRJNA252590 (accession number JOKJ00000000.1), respectively. Genomic comparisons between R1-200B4T and R2-400B4 revealed that these two isolates were very closely related, yet
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Pseudorhizobium pelagicum R1-200B4T (JOKI01000001) Neorhizobium galegae HAMBI 450T (HG938353) Shinella granuli CCUG 56671T (FM999739, FM999743) Ciceribacter lividus MSSRFBL1T (DSM 25528T) (KC189949, KP898900)
56
Ensifer adhaerens LMG 20216T (AJ505595, AM418746) Pararhizobium giardinii H152T (HQ394251, HQ394216)
58
Family Rhizobiaceae
Allorhizobium undicola LMG 11875T (EF457952, AM418784)
65
Agrobacterium radiobacter NCBI 9042T (ATCC 19358T) (HQ735079, HQ735092)
Rhizobium leguminosarum USDA 2370T (AJ294376, AJ294405) Phyllobacterium myrsinacearum ATCC 43590T (AJ294365, AJ294387) 51
90
Aminobacter aminovorans STM 2150 T (FR869646, AY785349) Pseudaminobacter salicylatoxidans KTC001T (NZ CAIU01000024)
60
Mesorhizobium loti LMG6125T (KM192346, KM192339) Aquamicrobium defluvii W13Z1 (|JENY01000009)
63 86
Family Phyllobacteriaceae
Nitratireductor aquibiodomus NL21T (NZ_BAMP01000152) 95
82
Balneimonas flocculans ATCC BAA-817T (JAEA01000006) Bosea thiooxidans LMG 26210T (FR871216, FR871211) Salinarimonas rosea DSM 21201T (NZ AUBC01000001) Nitrobacter winogradskyi Nb-255T (CP000115)
96
Rhodopseudomonas palustris HaA2 (CP000250)
99
Metalliresistens boonkerdii NS23T (EU177536, EU177520) 75
Oligotropha carboxidovorans OM5T (CP001196)
Family Bradyrhizobiaceae
Afipia felis ATCC 53690T (AGWZ01000001) Tardiphaga robiniae LMG 26467T (FR772688, FR873651) Bradyrhizobium japonicum USDA 6T (AP012206)
0.02 Fig. 2. Neighbor-joining phylogenetic tree based on partial concatenated sequences of recA and atpD genes (by this order and with a total of 820 positions) showing the relationships between Pseudorhizobium pelagicum gen. nov. sp. nov., the remaining genera from family Rhizobiaceae and the genera of the related families Phyllobacteriaceae and Bradyrhizobiaceae. The significance of each branch is indicated by a bootstrap value (in percentage) calculated for 1000 subsets (only values higher than 50% are indicated). Bar, 2 substitutions per 100 nucleotide positions.
distinct microorganisms. The analysis of nucleotide identity (ANI), based on whole genome sequencing and calculated using JSpecies software package v1.2.1 and default parameters [20], is high (99.98%) between the two strains over the core genome; however, coverage (98.37%) was not 100% as expected with clones of the same microorganism. This indicates that the genomes are not identical (i.e., clones) and contain genomic areas unique to one or the other strain. In fact, the R1-200B4T genome is slightly smaller (5.13 Mbp, 4539 CDS) compared to R2-400B4 (5.24 Mbp, 4619 CDS) with 168 unique genes between the two isolates. Most notably, R2400B4 contains a large lysogenic phage not present in R1-200B4T (Fig. S1). Furthermore, single nucleotide polymorphism (SNP) analysis between the two genomes revealed 1149 SNPs with a dN/dS ratio of 0.195, which provides additional evidence that these two isolates are not clones. Rather these data indicate that R1-200B4T and R2-400B4 belong to the same clonal frame, which describes bacterial lineages of common ancestry descent, in which replacement of genome fragments by recombination, selection, and drift by neutral genetic mutations has occurred [15]. In comparison, three non-clonal strains of Alteromonas macleodii comprising a single clonal frame (strains AltDE1, UM7/UM8, and UM4b) have a similarly high ANI (99.97%), yet exhibit only 87 SNPs and a maximum of 85 unique genes [14]. In addition to genomic comparisons, differences in antibiotic resistance (see below) and the RAPD patterns obtained with the primer M13 (5 -GAGGGTGGCGGTTCT-3 ) using the methodology described by Rivas et al. [21] confirmed that the two strains are not clones (Fig. S2). For comparison between the strains isolated in this study and their closest phylogenetic relatives, the 16S rRNA, recA and atpD gene sequences were identified from the R1-2004BT and R2-400B4 genomes. The 16S rRNA gene sequences of the two isolated strains were identical, thus only the sequence of the
type strain R1-200B4T was included in the phylogenetic analyses. Subsequent 16S rRNA, recA and atpD gene comparisons were made with those from GenBank using the BLASTN program [1] and the 16S rRNA gene sequences were also compared with those from EzTaxon-e server [11]. Sequences were aligned using the ClustalX software [30]. The distances were calculated according to Kimura’s two-parameter model [12]. Phylogenetic trees were inferred using the neighbour-joining (NJ) [24] and maximum likelihood (ML) [22] analyses. MEGA5 software [29] was used for all analyses. According to the results of comparison against the sequences held in EzTaxon-e server, the 16S rRNA gene sequence of strain R1-200B4T presents 97.0% and 95.6% similarity with respect to the type strains of the type species from genera Neorhizobium and Rhizobium, Neorhizobium galegae HAMBI 540T and Rhizobium leguminosarum USDA 2370T , respectively. The remaining genera of family Rhizobiaceae showed similarities lower than 95%. The NJ and ML phylogenetic analyses (Fig. 1 and Fig. S3, respectively) confirmed these results showing that the distance found between the strain R1-200B4T and N. galegae HAMBI 540T is similar or higher than those found among some species of family Phyllobacteriaceae previously mentioned in this work and among most of genera from Bradyrhizobiaceae. The recA and atpD genes have been sequenced in the type strains of the type species from all genera of family Rhizobiaceae and in most of type species of the families Phyllobacteriaceae and Bradyrhizobiaceae. Although for some species these sequences are available for strains other than the type they were included in this work. Since the sequences of recA and atpD genes are identical in the strains from this study, only the genes of the type strain R1-200B4T were included in the NJ and ML phylogenetic analyses that yielded phylogenetic trees with similar topology (Fig. 2 and Fig. S4, respectively). These analyses showed that strain R1-200B4T clustered with its
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Table 1A Calculated ANI values for available genomes of strains from type species of different genera from Family Rhizobiaceae. The accession numbers for the genomes of Pseudorhizobium pelagicum R1-200B4T , Neorhizobium galegae HAMBI 540T , Rhizobium leguminosarum 3841 and Ensifer adhaerens strain AT were, respectively, JOKI01000001, HG938353, AM236080 and NZ JNAE00000000, GCA 000421945, NZ JHXQ00000000, NZ ARBG00000000. Species
P. pelagicum R1-200B4T
N. galegae HAMBI 540T
R. leguminosarum 3841
E. adhaerens strain AT
A. undicola ORS 992T
A. radiobacter DSM 30147T
P. giardinii H152T
P. pelagicum R1-200B4T N. galegae HAMBI 540T R. leguminosarum 3841 E. adhaerens strain AT A. undicola ORS 992T A. radiobacter DSM 30147T P. giardinii H152T
100 75.9 74.0 73.6 72.4 73.8 73.2
100 75.1 73.3 72.6 73.6 73.5
100 74.0 72.7 73.3 74.6
100 73.0 72.6 76.1
100 72.7 72.2
100 72.6
100
closest relative N. galegae HAMBI 540T with 90% and 88.6% similarity values, respectively. The type strain of R. leguminosarum USDA 2370T was more distant with 87% and 86% similarity values in recA and atpD genes, respectively. However, similar values to those found between strain R1-200B4T and N. galegae HAMBI 540T were also found between R1-200B4T and Shinella granuli CCUG56671T with 90% and 88.8% similarity, respectively. These values are equal or lower than those presented by other members of Family Rhizobiaceae such as Ensifer adhaerens ATCC 33212T or S. granuli CCUG56671T and the recently described Ciceribacter lividus MSSRFBL1T with 90% and 93% similarity values, respectively, in recA and atpD genes. Also similar values, 91% and 93%, respectively, are found between E. adhaerens ATCC 33212T or S. granuli CCUG56671T . In families Phyllobacteriaceae and Bradyrhizobiaceae the closest related genera for which recA and atpD genes are availale presented similarity values near to 90% in both genes, as occurs between Aminobacter aminovorans STM 2150T and Pseudaminobacter salicylatoxidans KCT001T from family Phyllobacteriaceae or between Oligotropha carboxidovorans OM5T and Afipia felis ATCC 53690T from family Bradyrhizobiaceae. These values support those obtained from 16S rRNA gene analysis showing that the strain R1-200B4T belongs to a new genus within the family Rhizobiaceae. ANI values are commonly used to delineate species, but they have also been evaluated for genera delineation in a recent work where several new genera have been proposed finding that ANI values ranged from 75 to 80% among genera of the family Enterobacteriaceae [28]. The calculated ANI values between the genome of strain R1-200B4T and those of strains from family Rhizobiaceae available in databanks are in Tables 1A–1C. All of them are similar or lower than those found among genera of family Enterobacteriaceae. The calculated ANI value between the genomes of the strains R1-200B4T and N. galegae HAMBI 540T is 75.9%. This value is similar to the ANI values found between the strain N. galegae HAMBI 540T and R. etli CFN42T . These findings also support the classification of strains R1-200B4T and R2-400B4 in a new genus. The G + C content was calculated from the sequenced genome data being 62.8% for the strain R1-200B4T and 62.4% for the strain R2-400B4. This value is similar to those found in the remaining genera of Family Rhizobiaceae [13]. The cellular fatty acids were analysed by using the Microbial Identification System (MIDI; Microbial ID) Sherlock 6.1 and the library RTSBA6 according to the technical instructions provided by this system [25]. The strains were cultured aerobically on TY plates
[4] at 28 ◦ C and cells were collected during the late-exponential phase of growth. The obtained results showed that the major fatty acids of P. pelagicum are those from summed feature 8 (C18:1 6c/C18:1 7c) and the C16:0 , followed by C19:0 cyclo 8c, C8:1 7c 11-methyl, C18:0 , summed feature 3 (C16:1 6c/C16:1 7c) and summed feature 2 (C12:0 aldehyde?/C14:0 3OH/C16:1 iso I). Although some differences were found in the proportions of some fatty acids among genera from Family Rhizobiaceae (Table 2), the major fatty acids of this new genus coincide with those found in the remaining genera of the family Rhizobiaceae and also with those of families Phyllobacteriaceae and Bradyrhizobiaceae [31]. The phenotypic characterization was performed in this study using the methodology previously reported [19,23]. The type strains of the type species for all the defined genera in Family Rhizobiaceae were included in the phenotypic study as reference (Table 3). Phenotypic characteristics of the new species are reported below in the species description and the differences with respect to the type species of the genera from family Rhizobiaceae are recorded in Table 3. The two strains from this study differ in their natural antibiotic resistance; the strain R1-200B4T was sensitive to cefuroxime, whereas the strain R2-400B4 was resistant. A common characteristic of species from genus Rhizobium is their ability to form symbiotic relationships with plants, which in turn results in biological nitrogen fixation. This symbiosis requires, among others, three major genetic components: nodulation (nod), nitrogenase (nif) and nitrogen fixation (fix) genes. Initially nod genes are crucial in inducing communication factors and establishing symbiosis with the host [8]. Subsequently nitrogenase, an enzyme complex encoded by nifHDK genes, catalyzes nitrogen fixation with the assistance of more than 15 additional nif genes [27]. Since nitrogen fixation is an energy-consuming process, effective symbiosis also depends on the operation of a respiratory chain with a high affinity for O2 , closely coupled to ATP production. Under these circumstances, the regulation of oxygen is controlled by the fix gene family that codes for a special three-subunit terminal oxidase (cytochrome terminal oxidase, cbb3) and a membrane complex participating in electron transfer to nitrogenase [5]. In the case of R1-200B4T and R2-400B4, the genomes show no evidence of nod genes, suggesting that these two strains are free-living microorganisms that do not form symbiotic associations unlike many of their closest relatives. In addition, the absence of nif genes further indicates that these microorganisms do not perform nitrogen fixation, which requires nitrogenase. Although rhizobia
Table 1B Calculated ANI values for available genomes of strains from type species of different genera from Family Bradyrhizobiaceae. The accession numbers for the genomes of Bradyrhizobium japonicum USDA 6T , Afipia felis ATCC 53690T , Nitrobacter winogradskyi Nb-255T and Rhodopseudomonas palustris HaA2 were, respectively, NC 017249, NZ AGWZ00000000, NC 007406 and NC 007778. Species
B. japonicum USDA 6T
A. felis ATCC 53690T
N. winogradskyi Nb-25T
R. palustris HaA2
B. japonicum USDA 6T A. felis ATCC 53690T N. winogradskyi Nb-255 R. palustris HaA2
100 74.0 75.8 76.3
100 74.2 74.3
100 76.2
100
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Table 1C Calculated ANI values for available genomes of strains from type species of different genera from Family Phyllobacteriaceae. The accession numbers for the genomes of Mesorhizobium loti MAFF303099, Pseudaminobacter salicylatoxidans KCT001, Aquamicrobium defluvii W13Z1 were, respectively, NC 002678, NZ JAGL00000000, JENY00000000 and NZ BAMP00000000. Species
M. loti MAFF303099
P. salicylatoxidans KCT001
A. defluvii W13Z1
N. aquibiodomus NL21T
M. loti MAFF303099 P. salicylatoxidans KCT001 A. defluvii W13Z1 N. aquibiodomus NL21T
100 75.3 75.7 72.1
100 75.3 72.5
100 72.1
100
Table 2 Cellular fatty acid composition of the type species from genera of family Rhizobiaceae. Data are from this study. Fatty acids present in amounts lower than 1% are not shown. nd. not detected. Data are from this study. Fatty acid
P. pelagicum R1-200B4T
N. galegae HAMBI 540T
R. leguminosarum USDA 2370T
C. lividus DSM 25528T
E. adhaerens LMG 20216T
S. granuli DSM 18401T
A. radiobacter ATCC 19358T
A. undicola LMG 11875T
P. giardinii H152T
C16:0 C17:0 C18:0 C16:0 3OH C18:0 3OH C18:1 7c 11-methyl C19:0 cyclo 8c Summed feature 2 Summed feature 3 Summed feature 8
7.69 nd 2.26 nd 1.49 3.04 4.37 4.41 1.49 73.84
11.58 1.91 2.28 2.54 1.60 0.55 10.76 4.63 0.63 61.14
7.18 nd 12.71 nd 0.99 7.06 3.74 3.59 1.19 62.77
3.72 nd 8.88 nd 1.50 nd 8.06 3.13 nd 74.71
2.89 nd 4.05 1.75 2.52 2.09 0.42 6.03 0.94 79.31
10.68 nd 1.71 1.65 0.62 10.14 18.30 3.51 1.70 50.55
8.64 nd 1.08 4.82 0.42 0.37 2.86 6.10 2.06 73.64
0.88 nd 0.83 2.48 1.35 1.54 1.22 5.65 2.98 82.01
6.57 nd 0.62 nd 2.26 nd nd 8.77 6.89 74.89
Summed feature 2: (C14:0 3OH/C16:1 iso I/C12:0 aldehyde?). Summed feature 3: (C16:1 7c/C16:1 6c). Summed feature 8: (C18:1 7c/C18:1 6c).
are most commonly associated with plant symbiosis, it has been shown in numerous examples that this is not always the case [7]. Even within a given species, such as R. leguminosarum, some strains can lack the ability to form symbiotic relationships, while others are common symbiotic microorganisms [26]. More recently, Rhizobium sp. NT-26, isolated from a contaminated acid mine, was shown not to form nodules and lacked both nodulation and nitrogen fixation genes [3]. Nevertheless, as most genera of family Rhizobiaceae contain symbiotic and non-symbiotic species and it is possible that future species of the new genus Pseudorhizobium can be able to form nitrogen-fixing nodules in legumes, we have not included these characteristics in the genus or species descriptions.
Based on the phenotypic, chemotaxonomic and genotypic characteristics we propose to classify the studied strains into a novel genus named Pseudorhizobium gen. nov. and to a new species named P. pelagicum sp. nov. Description of genus Pseudorhizobium gen. nov. Pseudorhizobium (Gr. adj. pseudes false; N.L. masc. n. Pseudorhizobium, false Rhizobium). Aerobic, Gram negative non-spore forming rods forming white colonies on YMA. Optimal growth at 28 ◦ C and pH7. Catalase and
Table 3 Differential phenotypic characteristics of P. pelagicum and the type species from the remaining genera of family Rhizobiaceae. Data are from this study. +: positive, −: negative, w: weak. Characteristics
P. pelagicum R1-200B4T
N. galegae HAMBI 540T
R. leguminosarum USDA 2370T
C. lividus DSM 25528T
E. adhaerens LMG 20216T
S. granuli DSM 18401T
A. radiobacter ATCC 19358T
A. undicola LMG 11875T
P. giardinii H152T
Colony colour (on YMA)
White
Bluish black − + − +
Ivory
+ + + +
White– cream − − − −
White
Nitrate reduction Growth in 1% NaCl Growth in 7% NaCl Growth at 37 ◦ C
White– pink − + − +
+ + − +
+ + − +
White– cream + + − +
White– cream − + − +
White– cream − + − w
Assimilation of (API 20NE) Gluconate Malate
+ −
+ +
+ w
− +
− +
+ +
+ +
− +
− +
Assimilation of (API 32GN) l-Rhamnose d-Ribose Inositol Melibiose 5-Keto-gluconate 2-Keto-gluconate Propionate 3-Hydroxybutyrate l-Histidine l-Alanine l-Serine
+ − − − + w − − − w +
− + + + + − − − + + −
+ + + + − − − + + − −
+ + + − − − − − − − −
+ + + + − − + − + + +
+ + + − − − + + + + +
+ + + + w + + w + + −
+ w + − − − − − + + −
+ + + + − − − − + − −
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oxidase were positive. Nitrate reduction and aesculin hydrolysis were positive. Production of -galactosidase and urease was positive. The production of indol, arginine dehydrolase or gelatinase was negative. Growth was observed in presence of 7% NaCl. The main fatty acids are C18:1 7c/C18:1 6c. The G + C content of genomic DNA of the type strain of the type species is 62.8 mol%. Delineation of the genus was determined by the phylogenetic information from 16S rRNA gene sequence and by the analysis of the complete genome sequence. The type species is P. pelagicum. Description of Pseudorhizobium pelagicum sp. nov. P. pelagicum (pe.la.gi’cum. L. neut. adj. pelagicum of or belonging to the sea). Aerobic, Gram negative non-spore forming rods forming white colonies on YMA at 28 ◦ C that is the optimal growth temperature. Oxidase and catalase positive. Growth was obtained in a range from 10 to 37 ◦ C and pH 6 to 8. Weak growth observed at 40 ◦ C. The optimum pH is 7–7.5. No growth was observed at pH 4.5. Growth was observed in presence of 7% (w/v) NaCl. In API 20NE system nitrate reduction and aesculin hydrolysis were positive. Production of -galactosidase and urease was positive. The production of indol, arginine dehydrolase or gelatinase was negative. Glucose, l-arabinose, mannose, mannitol and gluconate were assimilated, but N-acetyl-glucosamine, malate, caproate, adipate, citrate and phenylacetate did not. The assimilation of maltose was weak. In API 32GN system the assimilation of l-rhamnose, N-acetyl-glucosamine, l-ribose, sucrose, maltose, mannitol, glucose, l-fucose, l-sorbose, l-arabinose, acetate, d,llactate, 5 keto-gluconate, l-serine and l-proline was positive. That of salicine, l-alanine, l-histidine, 2 keto-gluconate was weak. Inositol, melibiose, itaconate, suberate, malonate, glycogen, 3-hydroxybenzoate, 4-hydroxybenzoate, 3-hydroxybutyrate, propionate, caprate, valerate and citrate was negative. Sensitive to ciprofloxacin, oxitetracycline and gentamycin. Sensitive or weakly sensitive to neomycin. Resistant or weakly sensitive polymyxin B. Resistant to penicillin, ampicillin, erythromycin, and cloxacillin. Resistance to cefuroxime was variable. The fatty acid profile consisted of C18:1 6c/C18:1 7c (summed feature 8), C16:0 , C19:0 cyclo 8c, C8:1 7c 11-methyl, C18:0 , C16:1 6c/C16:1 7c (summed feature 3) and C12:0 aldehyde?/C14:0 3OH/C16:1 iso I (summed feature 2). The G + C content of the strain R1-200B4T is 62.8 mol%. No nodulation genes were found in the genome of the strains from this species. The type strain R1-200B4T (= LMG 28314T = CECT 8629T ) was isolated from Mediterranean seawater that was collected off the coast of Alicante, Spain. Acknowledgements This work was supported by funds from Junta de Castilla y León (Regional Spanish Government), MICINN (Central Spanish Government). “The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013.) under grant agreement no. 311975 for the MaCuMBA (http://www.macumbaproject.eu/) project”. MHRB is recipient of a JAE-Doc researcher contract from CSIC cofinanced by ERDF. JDFF is recipient of a PhD fellowship from Universidad de Salamanca. We thank Dr. J. Euzeby by his valuable help in provinding the correct ethymology for the name of the new taxon. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2004.08. 011
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Please cite this article in press as: N.E. Kimes, et al., Pseudorhizobium pelagicum gen. nov., sp. nov. isolated from a pelagic Mediterranean zone, Syst. Appl. Microbiol. (2015), http://dx.doi.org/10.1016/j.syapm.2015.05.003